LPG2 Gene Duplication in Leishmania infantum: A Case for CRISPR-Cas9 Gene Editing

Abstract

On the surface of the Leishmania promastigote, phosphoglycans (PG) such as lipophosphoglycan (LPG), proteophosphoglycan (PPG), free phosphoglycan polymers (PGs), and acid phosphatases (sAP), are dominant and contribute to the invasion and survival of Leishmania within the host cell by modulating macrophage signaling and intracellular trafficking. Phosphoglycan synthesis depends on the Golgi GDP-mannose transporter encoded by the LPG2 gene. Aiming to investigate the role of PG-containing molecules in Leishmania infantum infection process, herein we describe the generation and characterization of L. infantum LPG2-deficient parasites. This gene was unexpectedly identified as duplicated in the L. infantum genome, which impaired gene targeting using the conventional homologous recombination approach. This limitation was circumvented by the use of CRISPR/Cas9 technology. Knockout parasites were selected by agglutination assays using CA7AE antibodies followed by a lectin (RCA 120). Five clones were isolated and molecularly characterized, all revealing the expected edited genome, as well as the complete absence of LPG and PG-containing molecule expression. Finally, the deletion of LPG2 was found to impair the outcome of infection in human neutrophils, as demonstrated by a pronounced reduction (∼83%) in intracellular load compared to wild-type parasite infection. The results obtained herein reinforce the importance of LPG and other PGs as virulence factors in host-parasite interactions.

Major Molecular Factors Related to Leishmania Pathogenicity

“…Finally, it was discovered that deletion of LPG2 impairs the result of infection in human neutrophils, as evidenced by an 83 percent reduction in intracellular load compared to wild-type parasite infection. The findings support the role of LPG and other PGs in host-parasite interactions as virulence factors ( 35 ).…”

Targeted Deletion of Centrin in Leishmania braziliensis Using CRISPR-Cas9-Based Editing

“…Following the first successful attempt of gene replacement in L. major ( Cruz et al, 1993 ), genetic manipulation has now been achieved in different leishmania species. Since the recent introduction of CRISPR-Cas9-based methodologies, a significant advance has been made in this field: CRISPR-Cas-9 enabled the deletion of hundreds of genes, including, BTN1 ( Ishemgulova et al, 2018 ) and LeishIF4E-3 ( Shrivastava et al, 2019 ), genes encoding protein kinases ( Baker et al, 2021 ) and flagellar proteins ( Beneke et al, 2019 ) in L. mexicana , as well as LPG2 in L. infantum ( Jesus-Santos et al, 2020 ), RAD51 in L. major ( Damasceno et al, 2020 ) and Ros3 in L. braziliensis ( Espada et al, 2021 ). Herein, we have used the LeishGEdit tool box ( Beneke et al, 2017 ; Beneke and Gluenz, 2019 ) to manipulate the L. braziliensis genome.…”

Construction of PX-LmGP63 Using CRISPR-Cas9 as Primary Goal for GP63 gene Knockout in Leishmania major and Leishmanization

“…The results of their study strengthen the importance of LPG and other PGs as virulence parameters in host-parasite interactions (28). The CRISPR-Cas9 system uniquely enables the simultaneous targeting of multiple genome sites (29).…”

“…In this study, con-structed pX-leish was created with three features: (1) Promoters congenial with Leishmania parasites; (2) inserting antibiotic selection marker; (3) all-in-one vector designing, including all components required for CRISPR/Cas9. Also, in a similar study, Jesus-Santos et al in 2020 showed that the deletion of LPG2 impaired the outcome of infection in human neutrophils, as presented by a pronounced diminution (~83%) in intracellular load compared to wildtype parasite infection (28).…”